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Researchers in Germany have developed a new cell-based platform they say can be used to accurately identify antigens responsible for triggering the activation of cytotoxic CD8+ T cells in autoimmune disorders, infectious diseases, and cancer. The technique uses an engineered T-cell line that carries T-cell receptor (TCR) molecules that are present on the patients’ own CD8+ T cells, and which fluoresce when bound to their activating peptide antigens. These cells are then incubated with antigen-presenting cells (APCs) engineered to display a plasmid-encoded combinatorial peptide library. Fluorescence of individual T cells indicates that the cell has been activated by a peptide expressed on an APC to which it comes into contact.

Because the APCs are engineered to display short peptides, the approach doesn’t require that the cells carry out antigen processing. Instead, T-cell fluorescence identifies those peptide sequences from the library that represent the T-cell-activating mimotopes of native antigens that would be presented by MHC class I molecules on APCs in vivo. Moreover, claim the Ludwig Maximilians University, and Max Planck Institute of Neurobiology researchers, the technique is incredibly sensitive because even a single activated (and thus fluorescing) T cell can be spotted. The antigen-expressing plasmid can then be recovered from the APC, amplified, and analyzed to identify the peptide.

And as the method layers the APCs on top of the T cells, the two cell types remain in contact through gravity, so TCR receptor affinity isn’t an issue either. Klaus Dornmair, Ph.D., and colleagues report on their technique in Nature Medicine in a paper titled “Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides.”

The researchers co-transfected the T hybridoma cell line 58α−β− (which lacks endogenous TCR α and β chains) with the human influenza-specific TCR JM22, human CD8 α and β chains, and super green fluorescent protein (sGFP) under the control of the nuclear factor of activated T cells (NFAT). For the test cases the α and β chains were derived from T cell clones, but they could have originated from T cells isolated from patient tissue, the researchers explain.

In an initial set of experiments the T cells were overlaid with engineered COS-7 cells that had been stably transfected with HLA-A (COS-A2stable), and the synthetic peptide flu(58-66) added to the mix. This process led to about 50% of the T cells fluorescing when analyzed using flow cytometry. In contrast, when COS-7 cells lacking HLA-A2 were used, or the flu(58-66) peptide was replaced with an irrelevant cytomegalovirus-derived peptide, none of the 250,000-plus 58-JM22-CD8-sGFP cells fluoresced.

The team then introduced the sequence coding for flu(58-66) into an expression plasmid, and supertransfected the COS-A2stable cells with the construct. When the resulting flu(58-66)-expressing cells were overlaid onto the 58-JM22-CD8-sGFP, about 35% of the reporter T cells were activated, not a huge proportion less than when the synthetic flu(58-66) was used instead. “This experiment shows that short peptides encoded by pcDNA plasmids may be transported efficiently into the lumen of the endoplasmic reticula of COS-7 cells, where they are loaded onto class I MHC molecules,” the authors write.

Generating stable HLA-transfected cells is labo­rious and time-consuming, however, so in an attempt to negate this requirement, the researchers co-transfected an HLA-A2 sequence-carrying plasmid (RSV-A2) and a flu(58-66) plasmid transiently into COS-7 cells (COS-7-A2trans), and 32 hours later added the 58-JM22-CD8-sGFP cells. Even using this procedure, 11–17% of the 58-JM22-CD8-sGFP cells were activated and fluorescence detected. TCR activation was also specific, as the 58-JM22-CD8-sGFP cells weren’t activated by COS-7empty cells transfected with the flu(58–66) plasmid, or by COS-7-A2trans cells transfected with an empty (i.e., no flu sequence) plasmid. “Inhibition of the intracellular transport of HLA molecules with brefeldin A also abrogated the activation of 58-JM22-CD8-sGFP cells,” the researchers add.

In a final set of tests, the team generated combinatorial peptide libraries in a plasmid, and stably co-transfected the plasmid with RSV-A2 into COS-7 cells. When these cells were overlaid on the 58-JM22-CD8-sGFP reporter cells, individual fluorescing T cells that were in direct contact with COS-7 cells could be identified using fluorescence microscopy. These cells were then isolated together with the subjacent COS-7 cells using a capillary pipette, and the peptide-containing plasmids in the COS-7 cells subcloned and their sequences determined.

In fact sequence analysis of the plasmid sequences identified five mimotopes of flu-(58-66) that were different from the parent sequence but had still activated the T cells. And when the experiment was repeated another 12 times, a total of nine mimotopes were identified in 123 isolated COS-7 cells. For a number of these mimotopes, search of a nonredundant protein database comprising all species of homologous peptides enabled identification of the influenza matrix protein as the candidate parent antigen. “The identification of several mimotopes of the model TCR JM22 proves that our approach is suitable for discovering previously unknown T cell antigens,” the investigators state.

They say their technique could be applied for the unbiased identification of antigenic mimotopes that are recognized by CD8+ T cells such as autoaggressive T cells in autoimmune diseases, tumor-infiltrating T cells in cancers, and antiviral and antibacterial T cells in infectious diseases. “This method is extremely sensitive because single cells serve as readouts, and several million library peptides can be examined within a few hours,” the authors stress. “Precise knowledge of T cell target antigens will facilitate the development of biomarkers for the detection of viral infections and tumors, assist in the diagnosis of autoimmune diseases and offer new therapeutic options, including vaccinations against viral infections or tumors with new mimotopes or TCR gene therapy.”

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